Throughout this application, various references are cited in square brackets to describe more fully the state of the art to which this invention pertains. The disclosure of these references is hereby incorporated by reference into the present disclosure.
Oxidative stress (OS) is a biological state that occurs when a cell's antioxidant capacity is overwhelmed by reactive oxygen species (ROS), causing a redox imbalance. Reactive oxygen species are a type of free radical, which is formed with oxygen. Free radicals are chemical substances that contain one or more unpaired orbital electrons and are therefore unstable and liable to react with other molecules to form more stable compounds with a lower energy state. In an attempt to achieve this stable state, ROS reacts with proteins, lipids, and DNA within the cell. This can result in damage and even inactivation of cellular components such as enzymes, membranes, and DNA. As such, ROS and oxidative stress as a whole have been suggested to participate in the initiation and/or propagation of diseases such as cardiovascular and inflammatory diseases, cancer, and diabetes. [Valko M, et al. Mol. Cell. Biochem. (2004) 266:37]
ROS can be produced on a regular basis during oxidative metabolism and in more potent levels during inflammatory processes. During oxidative metabolism, electrons are lost from the electron transport chain and combine with oxygen, resulting in the formation of superoxide anion (O2—). [Boveris A, Chance B. Biochem. J. (1973)134:707] At the time of inflammation, macrophages and neutrophils that contain the NADPH oxidase complex generate superoxide radicals and hydrogen peroxide to aid in the destruction of foreign agents. [Rosen G M, et al. FASEB J. (1995) 9:200] Environmental factors such as tobacco smoke, UV radiation, exposure to atmospheric oxygen, overexertion during exercise, and the consumption of alcohol and certain foods can also result in the generation of ROS. [Bunker V W. Med. Lab. Sci. (1992) 49:299; Powers S K, Jackson M J. Physiol. Rev. (2008) 88:1243] Though many of these factors can be avoided or limited, as humans, our omnivorous diet exposes us to a variety of foods, some of which may contribute to increased oxidative stress in the gut. An uncontrolled increase of ROS in the gastrointestinal mucosa can lead to inflammatory or ischemic disorders. [Parks D A, et al. Surgery (1983) 94:415; Thomson A, et al. Dig. Dis. (1998) 16:152] Oxidative stress has been postulated to play a role in inflammatory bowel disease (IBD) initiation and progression. The binding of an inflammatory stimulus to its cellular receptor triggers the activation of specific intracellular signaling pathways to upregulate the production of inflammatory mediators. Therefore, antioxidative stress mechanisms and antioxidants are key to limiting the proliferation of ROS and re-establishing a stable redox balance.
ROS, bacterial cell wall lipopolysaccharide (LPS) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) can trigger the activation of multiple signaling pathways including the phosphofylation cascades leading to the activation of mitogen activated protein kinase (MAPKs) and nuclear factor B (NF-κB). [Aggarwal B B. Nat. Rev. Immunol. (2003) 3:745; Tak P P, J. Clin. lnvest. (2001) 107:7] LPS binding to TLR4 and TNF-α to the TNF receptor activate the Ikappa B kinase (IKK)-NF-κB pathway and the three MAPK pathways: ERK 1/2, JNK, and p38. These pathways in turn activate a variety of transcription factors that include NF-κB and activator protein-1 (AP-1). [Dalton T P, et al. Annu. Rev. Pharmacol. Toxicol. (1999) 39:67; Morel Y, Barouki R. Biochem. J. (1999) 342 Pt 3:481] AP-1 binding sites are present in the promotor regions of a large number of proinflammatory cytokine and adhesion molecule genes [Dalton T P, et al. Annu. Rev. Pharmacol. Toxicol. (1999) 39:67; Morel Y, Barouki R. Biochem. J. (1999) 342 Pt 3:481] and play a role in regulating the expression of γ-GCS, the key enzyme in the synthesis of glutathione (GSH). [Rahman I, et al. FEBS Lett. (1998) 427:129] Hydrogen peroxide is able to permeate the cell membrane and act as a cell-signaling molecule by oxidizing the thiol moiety of sulfhydryl-containing proteins involved in signaling transduction pathways [Dalton T P, et al. Annu. Rev. Pharmacol. Toxicol. (1999) 39:67; Morel Y, Barouki R. Biochem. J. (1999) 342 Pt 3:481; Arrigo A P. Free Radic. Biol. Med. (1999) 27:936]. Both NF-κB and AP-1 are redox-sensitive and are both activated during oxidative stress and inflammation. Inhibiting their expression and resulting activity, are key to limiting the expression of oxidative stress and inflammatory mediators.
Aerobic organisms can increase production of biochemical antioxidants such as glutathione (GSH) and induce endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase, thioredoxin reductase (TrxR), glutathione reductase (GR) and glutathione peroxidase (GPx) to inactivate oxidants, forming instead biologically inert products. [Cimino F, et al. Curr. Top. Cell. Regul. (1997) 35:123; Halliwell B. Free Radic. Res. Commun. (1990) 9:1] GSH (γ-glutamylcysteinylglycine) is the major non-enzymatic regulator of redox homeostatis and is ubiquitously present in all cell types. [Meister A, Anderson M E. Annu. Rev. Biochem. (1983) 52:711] It can directly scavenge free radicals or act as a substrate for GPx and glutathione S-transferase (GST) during detoxification of hydrogen peroxide, lipid hydroperoxides and electrophilic compounds. Glutathione is synthesized in two sequential ATP-dependent reactions catalyzed by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GS). [Griffith O W. Free Radic. Biol. Med. (1999) 27:922] Recently, food-derived compounds like curcumin and flavonoids have been shown to up-regulate intracellular GSH synthesis. [Biswas S K, et al. Antioxid. Redox Signal (2005) 7:32; Myhrstad M C, et al. Free Radic. Biol. Med. (2002) 32:386] In addition, olive oil biophenols also influence the increase in GPx and GR antioxidant enzyme activities. [Yang HyeKyung, J. Agric. Food Chem. (2005) 53:4182] This demonstrates the role of food-based components in influencing our bodies' intracellular antioxidant defense systems.
A variety of egg components have been cited to possess antimicrobial, antiadhesive, immunomodulatory, anticancer, antihypertensive, and antioxidant activities; behave as protease inhibitors; increase nutrient bioavailability; and provide a source of functional lipids. [Kovacs-Nolan J, et al. J. Agric. Food Chem. (2005) 53:8421] If biologically active properties can be separated into those belonging to the egg white versus the egg yolk, the majority of bioactive peptides thus far have been found in the egg white. This is in part due to the higher percentage composition of egg white (w/v) [Cotterill O J, Geiger G S. Poult. Sci. (1977) 56:1027] and increased variety of egg white proteins compared to that of the yolk. [Sugino H, et al. In: Yamamoto T, Juneja L R, Hatta H, Kim M, editors. Hen Eggs, Their Basic and Applied Science New York: CRC Press; (1997) p. 13].
A variety of egg yolk peptides have been shown to have bioactive qualities. Lipoprotein (LDL) peptides are antimicrobial [Brady D, et al. J. Food Sci. (2003) 68:1433] and sialyiglycopeptides are antiadhesive. [Sugita-Konishi Y, J. Agric. Food Chem. (2002) 50:3607] Phosvitin, an iron-binding highly phosphorylated protein in the egg yolk, was found to have a high affinity for binding calcium, thereby increasing the bioavailability of this nutrient [Jiang B, Mine Y. Biosci. Biotechnol. Biochem. (2001) 65(5)1187] Phosvitin phosphopeptides have also been noted to have antioxidant and more recently, antioxidative stress properties [Katayama S, et al. J. Agric. Food Chem. (2006) 54:773; Katayama S, et al. J. Agric. Food Chem. (2007) 55:2829]
Egg yolk phosvitin is a highly phosphorylated protein with 10% phosphorus monoesterified to 57.5% serine (Ser) residues [Allerton S E, Perlmann G E. J. Biol. Chem. (1965) 240:3892; Taborsky G. Adv. Inorg. Biochem. (1983) 5:235] Oligophosphopeptides (PPPs) prepared from egg yolk phosvitin with 35% phosphate retention, have enhanced calcium- and iron-binding abilities, thereby fulfulling a potential role in increasing their uptake in the intestinal tract. [Jiang B, Mine Y. Biosci. Biotechnol. Biochem. (2001) 65:1187; Jiang B, Mine Y. J. Agric. Food Chem. (2000) 48:990; Feng FengQin, Mine Y. Int. J. Food Sci. Tech. (2006) 41:455] It was previously shown that phosvitin upon alkaline hydrolysis, enzymatic cleavage, and liquid chromatographic separation, resulted in an oligophosphopeptide fraction with antioxidative stress properties. [Katayama S, at al. J. Agric. Food Chem. (2006) 54:773] However, the procedure for obtaining this phosvitin-derived oligophosphopetide fraction utilized non-GRAS (generally recognized as safe) chemicals, was time-consuming and was not “industry-friendly” for scaling up purposes.
It would be desirable, thus, to isolate peptides and compositions capable of inducing an antioxidative response. It would also be desirable to develop a method of preparing and isolating antioxidant peptides and compositions comprising said peptides from a natural source such as egg yolk that use ingredients that are generally recognized as safe (GRAS) for use in food.
The Applicant has now identified novel peptides and compositions capable of inducing an antioxidative response.